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  • 94
    Jena Bioscience mant gtp
    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than <t>GTP/GDP</t> interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
    Mant Gtp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gtp
    Nuclear transport factors are be introduced to nuclear periphery using digitonin permeabilization of the plasma membrane. A) Cells were incubated with R-phycoerythrin after the digitonin permeabilization protocol was performed with (left) 0 µg/mL digitonin, for which no R-phycoerythrin is detected in the cell, (middle) 34 µg/mL, for which R-phycoerythrin has entered the cytosol but is excluded from the nucleus, and (right) 100 µg/mL digitonin for which R-phycoerythrin is seen throughout the cytosol and nucleus. (Scale Bar = 10 µm). B-C) The Coomassie-stained gels show purified Ran, NTF2, Kap1-α, Kap1- β proteins with minimal contaminants. D) Cells were incubated with an ATP-regenerating system, <t>Ran-GTP,</t> NTF2, and an <t>NLS-TD-Tomato</t> cargo in the presence and absence of Kap1-α and Kap1- β . E) Amount of NLS-TD-Tomato accumulated in the nucleus was measured by quantifying the average fluorescence signal in the nucleus, as defined via Hoechst staining. (n=5 cells, error bars represent standard deviation). F-G) Removal of kaps was confirmed with quantitative IF. (Scale Bar = 10 µm; error bars represent standard deviation; n=5 cells) .
    Gtp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 628 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gtp
    Cyclic dinucleotides activate the inflammasome in THP-1 cells and murine BMMs. ( A ) PMA-differentiated THP-1 cells (left panel) were treated with ATP (5 mM; 6 h) or transfected (Lipofectamine 2000; lpftm) with c-diGMP (cdG; 5 nmol; 6 h) or poly(dA:dT) (dA:dT; 5 μg/ml; 6 h). LPS-primed BMMs (right panel) were treated with ATP (5 mM; 6 h) or transfected (X-tremegene HP; Xtrm) with c-diGMP (10 nmol; 6 h), <t>c-diAMP</t> (10 nmol; 6 h), cGMP (10 nmol; 6 h) or <t>GTP</t> (10 nmol; 6 h). Supernatants were immunoblotted for immature IL-1β (pro-IL1β), active IL-1β (p17), immature caspase-1 (pro-Cas1) or active caspase-1 (Cas1, p20 and p10, respectively). ( B ) IL-1β levels were measured by ELISA (BD Biosciences) in 6 h supernatants of LPS-primed BMMs after the indicated treatment with c-diGMP or c-diAMP, as in A . ( C ) IL-1β levels were measured by ELISA in BMMs infected with IPTG-induced L. pneumophila ]. Graphs present means±standard error from three independent experiments. BMMs, bone marrow-derived macrophages; c-diAMP, 3′-5′ diadenylate; c-diGMP, 3′-5′ diguanylate; ELISA, enzyme-linked immunosorbent assay; MOI, multiplicity of infection.
    Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1716 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare α 32 p gtp
    Analysis of functionally active pol III transcription complexes separated by a glycerol gradient. Several identical transcription reactions were performed in a volume of 25 µl each as described in Materials and Methods. After incubation they were pooled. Aliquots of 200 µl from this pool (corresponding to eight transcription reactions) were applied to a 4.2 ml glycerol gradient and centrifuged for 3 h at 50 000 r.p.m. in an SW 60 rotor. After centrifugation, fractions of 350 µl were collected. The corresponding fractions from six parallel gradients were pooled. ( A ) In vitro transcription. The fractions from the gradients and the load fraction were incubated with nucleotides, [α- 32 <t>P]GTP</t> and RNase Block as described above. No additional DNA was added. After incubation, the RNA in the samples was extracted as described above and loaded onto a 6% denaturing polyacrylamide gel. Lane 1, 25 µl load fraction; lanes 2–13, 50 µl glycerol gradient fractions. Fraction 1 is the top fraction ( B ) Western blot. The remaining pooled fractions were treated with Strataclean Resin (Stratagene) and loaded onto a 12.5% SDS gel. After blotting, the membrane was incubated with monoclonal anti-TBP antibodies and subsequently incubated with [ 125 I]anti-mouse antibodies as secondary antibodies. The membrane was analysed with a phosphorimager. The membrane was not stripped, but incubated with a mixture of SW5 and 3B9 antibodies and subsequently incubated with 125 I-labelled anti-mouse antibodies. The final analysis was performed with a phosphorimager and by autoradiography. Note that the band in fraction 8 at the height of the La signal was already visible after TBP incubation (data not shown). It is presumably an artefact due to the second antibody. Lane 1, 160 µl of load fraction; lanes 2–13, 1.85 ml glycerol gradient fractions; lane 14, 5 µl IIIBβ; lane 15, 5 µl of a La fraction, purified from PCA (20 ng La/µl). ( C ) In vitro transcription. The in vitro transcription was performed as described in (A) using a mixture of fractions 8 and 9 of a typical gradient. To ensure single round conditions in lanes 6–10, heparin was added at a final concentration of 600 µg/ml. Lanes 1–5, incubation without heparin for 2, 5, 10, 20 and 40 min; lanes 6–10, incubation with heparin for 2, 5, 10, 20 and 40 min. ( D ) Multiple round transcription of fractions 8 and 9 for 60 min. The gel was autoradiographed for a shorter time period as in (C). BSA (10 µg) was added to the reaction in order to avoid any protein effects. Lane 1, no additional La; lanes 2–4: addition of 15, 30 or 60 ng recombinant La.
    α 32 P Gtp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore gtp γ s
    Capacity of <t>GTP</t> <t>γ</t> S to Inhibit PTH Radioligand Binding to Wild-Type and Mutant PTHRs in COS-7 Cell Membranes
    Gtp γ S, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cytoskeleton Inc gtp bound rhoa
    Organization of actin stress fiber, the association of actin with paxillin, and <t>RhoA</t> activity are decreased in BHDS lung fibroblasts. (A) Representative confocal images of phalloidin and paxillin staining in lung fibroblasts are shown; the results of BHDS lung fibroblasts isolated from patient 2 harboring c.769_777del TCC (exon 7) mutation are presented. The immunofluorescence staining was performed in both control ( n = 4) and BHDS ( n = 4: patients 2, 7, 10, and 11). Scale bars: 50 μm in each panel. (B) The levels of <t>GTP</t> ‐bound RhoA (Rho‐ GTP ) and Rho GTP /total RhoA ratio are decreased in BHDS lung fibroblasts ( n = 4: patients 2, 7, 10, and 11) as compared with those of control lung fibroblasts ( n = 4) (* P
    Gtp Bound Rhoa, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 91/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    GE Healthcare m7 gtp sepharose beads
    Full-length hAGO2 binds to m 7 <t>GTP-Sepharose</t> nonspecifically in in vitro pull-down experiments and at low levels, indicating background binding. ( A ) Pull-down experiments using recombinant GST-eIF4E and hexahistidine-SUMO-tagged hAGO2 MID domain with m
    M7 Gtp Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 98/100, based on 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher mant gtp
    DOCK10 is a <t>GEF</t> for Rac1 and Cdc42 in vitro and in cells. (A, B) In vitro GEF assay performed on Cdc42 (A) and Rac1 (B), using GST-DHR2 domains of DOCK9, DOCK10, and DOCK11. GST-Dbl and GST-Trio GEF1 were used as positive control GEFs for Cdc42 and Rac1, respectively, whereas GST alone was used as negative control. Results are expressed as relative fluorescence units (RFU) vs. time. (C–E) Active GTPase pull-down assays. <t>GTP-loaded</t> Cdc42 (C) or GTP-loaded Rac1 (D, E) were pulled down from protein lysates of HEK293 cells expressing wild-type pEGFP-DOCK9, -10 or -11 constructs or DOCK10 GD. GFP vector was used as a negative control. Shown are representative experiments. GTP-bound and total Cdc42 or Rac1 were detected by Western blot, using anti-Cdc42 and anti-Rac1 antibodies, respectively. Protein expression in the cell lysates was verified by immunoblotting with an anti-GFP antibody. Histograms underneath each Western blot show the quantification of the corresponding GTPase activation assays. The ratio of GTP-Rac1 (or Cdc42) over total Rac1 (or Cdc42) was calculated from at least three independent experiments. Error bars indicate SEM, ** p
    Mant Gtp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore guanosine triphosphate gtp
    Combination of CTP and <t>GTP</t> with <t>2CMC</t> in the Huh7-p6-luc model (A) and the Huh7-p6 model (B). The cells were treated with 2CMC, CTP or GTP, alone or in combination for 72 h before measurement of luciferase activity. Data are the mean ± SEM of four to six independent experiments. *, P
    Guanosine Triphosphate Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim gtp
    Combination of CTP and <t>GTP</t> with <t>2CMC</t> in the Huh7-p6-luc model (A) and the Huh7-p6 model (B). The cells were treated with 2CMC, CTP or GTP, alone or in combination for 72 h before measurement of luciferase activity. Data are the mean ± SEM of four to six independent experiments. *, P
    Gtp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cytoskeleton Inc gtp
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Gtp, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 93/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Jena Bioscience 6 thio gtp
    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. <t>GTP-bound</t> Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by <t>6-Thio-GTP.</t> Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    6 Thio Gtp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    NewEast Biosciences rac1 gtp
    HGF-induced activation of <t>Rac1</t> and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or <t>GTP-Rac1</t> (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.
    Rac1 Gtp, supplied by NewEast Biosciences, used in various techniques. Bioz Stars score: 89/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare m7 gtp sepharose 4b
    pUL69 associates with polysomes. MRC5 cells were harvested at 72 h after infection with TN wt . ( A ) Polysome preparation and analysis. Cytoplasmic extracts of infected MRC5 cells were prepared and fractionated by centrifugation on a 10 to 50% sucrose gradient. The bottom of gradient is to the left, and fractions 1 to 4 correspond to polysomes. ( Top ) UV absorbance profile of gradient fractions; ( Middle ) Analysis of total RNA present in gradient fractions by electrophoresis in an <t>agarose</t> gel; ( Bottom ) Western blot analysis of proteins in gradient fractions using antibodies for pUL69 and PABPC1. ( B ) Disruption of polysomes displaces pUL69 from rapidly sedimenting fractions. Cytoplasmic extracts were treated with EDTA (+) or left untreated (−) and subjected to centrifugation; proteins in fractions 1 to 4 containing polysomes were precipitated and analyzed by Western blot by using antibodies for pUL69, PABPC1 and eIF4A1. T, total cytoplasmic extract. ( C ) pUL69 interacts with viral mRNAs. Cytoplasmic extracts were subjected to immunoprecipitation by using an antibody to pUL69 pUL84 or they were mixed with protein A/G <t>Sepharose</t> without first receiving a primary antibody (No Ab). mRNA was isolated from the immunoprecipitates and quantified by RT-PCR using sequence-specific primers.
    M7 Gtp Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    VANGL2 LTD gtp binding protein adaptor protein complex
    Proposed model. ( A ) Model depicting Arfrp1- and AP-1-mediated TGN sorting of <t>Vangl2.</t> Arfrp1 is recruited to TGN membranes upon <t>GTP</t> binding, possibly mediated by a TGN localized GEF. Subsequently, GTP-bound Arfrp1 recruits AP-1 to TGN membranes. GTP-bound Arfrp1 also promotes an open conformation of AP-1 that directly interacts with the tyrosine motif on Vangl2 cytosolic domain, thereby enriching Vangl2 in budding vesicles. Binding of Vangl2 cytosolic domain to AP-1, in turn, stabilizes the membrane association of AP-1 to allow sufficient time for AP-1 polymerization (possibly with clathrin as a coat outer layer) and vesicle budding. This model is consistent with reports showing that tyrosine sorting motifs promote membrane recruitment of AP-1 mediated by Arf1 ( Crottet et al., 2002 ; Lee et al., 2008 ). ( B ) The asymmetrically localized PCP signaling molecules, including Vangl2 and Frizzled 6, are sorted by different sorting machineries for export from the TGN. Differential TGN sorting and polarized trafficking of these signaling receptors may contribute to their asymmetric distribution and the laterally polarized organization of epithelial cells. DOI: http://dx.doi.org/10.7554/eLife.00160.017
    Gtp Binding Protein Adaptor Protein Complex, supplied by VANGL2 LTD, used in various techniques. Bioz Stars score: 88/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than GTP/GDP interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than GTP/GDP interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques:

    ITC analysis. The upper and lower panels display the ITC titration curves and the binding isotherms, respectively, for MM1309 with GTP·Mg 2+ ( a ), GTP without Mg 2+ ( b ), GDP·Mg 2+ ( c ), and GMPPNP·Mg 2+ ( d ). N , the binding stoichiometry; K b , the observed binding constant; K d ( K d = 1/ K b ), the dissociation constant; ∆ H , the binding enthalpy; ∆ S , the binding entropy

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: ITC analysis. The upper and lower panels display the ITC titration curves and the binding isotherms, respectively, for MM1309 with GTP·Mg 2+ ( a ), GTP without Mg 2+ ( b ), GDP·Mg 2+ ( c ), and GMPPNP·Mg 2+ ( d ). N , the binding stoichiometry; K b , the observed binding constant; K d ( K d = 1/ K b ), the dissociation constant; ∆ H , the binding enthalpy; ∆ S , the binding entropy

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques: Titration, Binding Assay

    Stereo views of the GTP binding sites. a The bound GMPPNP molecule in the T. aquaticus EF-Tu·GMPPNP·Mg 2+ structure. b The bound GMPPNP molecule in the MM1309·GMPPNP·Mg 2+ structure. The F o – F c omit map (contoured at 3.3 σ) of the bound GMPPNP·Mg 2+ in the MM1309 active site. c , d Close-up stereo views around the γ-phosphate group of the bound GMPPNP in T. aquaticus EF-Tu·GMPPNP·Mg 2+ ( c ) and MM1309·GMPPNP·Mg 2+ ( d ). The amino acid residues surrounding the phosphate groups and the magnesium ions of the bound GMPPNP·Mg 2+ are depicted by stick models. e The bound GDP molecule in the MM1309·GDP structure. The F o – F c omit map (contoured at 4.0 σ) of the bound GDP·Mg 2+ in the MM1309 active site. f The GTP binding site in the MM1309 apo form. The MM1309 residues that are located close to the bound guanine nucleotide are represented as stick models. The P-loop motifs (Gly17–Thr25 in EF-Tu and Gly7–Thr15 in MM1309) are shown in sky blue . The switch I regions are colored pink . Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: Stereo views of the GTP binding sites. a The bound GMPPNP molecule in the T. aquaticus EF-Tu·GMPPNP·Mg 2+ structure. b The bound GMPPNP molecule in the MM1309·GMPPNP·Mg 2+ structure. The F o – F c omit map (contoured at 3.3 σ) of the bound GMPPNP·Mg 2+ in the MM1309 active site. c , d Close-up stereo views around the γ-phosphate group of the bound GMPPNP in T. aquaticus EF-Tu·GMPPNP·Mg 2+ ( c ) and MM1309·GMPPNP·Mg 2+ ( d ). The amino acid residues surrounding the phosphate groups and the magnesium ions of the bound GMPPNP·Mg 2+ are depicted by stick models. e The bound GDP molecule in the MM1309·GDP structure. The F o – F c omit map (contoured at 4.0 σ) of the bound GDP·Mg 2+ in the MM1309 active site. f The GTP binding site in the MM1309 apo form. The MM1309 residues that are located close to the bound guanine nucleotide are represented as stick models. The P-loop motifs (Gly17–Thr25 in EF-Tu and Gly7–Thr15 in MM1309) are shown in sky blue . The switch I regions are colored pink . Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques: Binding Assay

    Superposition of MM1309 with EF-Tu, aSelB, and aIF2γ, represented by ribbon models. a Superposition of the MM1309 structures in the GMPPNP-bound, GDP-bound, and apo forms. b Superposition of MM1309 with T. aquaticus EF-Tu in the GTP-bound form (PDB code: 1TTT). c Superposition of MM1309 with T. aquaticus EF-Tu in the GDP-bound form (PDB code: 1TUI). d Superposition of MM1309 with M. maripaludis aSelB (PDB code: 4ACA) and with P. abyssi aIF2γ (PDB code: 1KK0)

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: Superposition of MM1309 with EF-Tu, aSelB, and aIF2γ, represented by ribbon models. a Superposition of the MM1309 structures in the GMPPNP-bound, GDP-bound, and apo forms. b Superposition of MM1309 with T. aquaticus EF-Tu in the GTP-bound form (PDB code: 1TTT). c Superposition of MM1309 with T. aquaticus EF-Tu in the GDP-bound form (PDB code: 1TUI). d Superposition of MM1309 with M. maripaludis aSelB (PDB code: 4ACA) and with P. abyssi aIF2γ (PDB code: 1KK0)

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques:

    Nuclear transport factors are be introduced to nuclear periphery using digitonin permeabilization of the plasma membrane. A) Cells were incubated with R-phycoerythrin after the digitonin permeabilization protocol was performed with (left) 0 µg/mL digitonin, for which no R-phycoerythrin is detected in the cell, (middle) 34 µg/mL, for which R-phycoerythrin has entered the cytosol but is excluded from the nucleus, and (right) 100 µg/mL digitonin for which R-phycoerythrin is seen throughout the cytosol and nucleus. (Scale Bar = 10 µm). B-C) The Coomassie-stained gels show purified Ran, NTF2, Kap1-α, Kap1- β proteins with minimal contaminants. D) Cells were incubated with an ATP-regenerating system, Ran-GTP, NTF2, and an NLS-TD-Tomato cargo in the presence and absence of Kap1-α and Kap1- β . E) Amount of NLS-TD-Tomato accumulated in the nucleus was measured by quantifying the average fluorescence signal in the nucleus, as defined via Hoechst staining. (n=5 cells, error bars represent standard deviation). F-G) Removal of kaps was confirmed with quantitative IF. (Scale Bar = 10 µm; error bars represent standard deviation; n=5 cells) .

    Journal: bioRxiv

    Article Title: Cargo modulates the conformation of the nuclear pore in living cells

    doi: 10.1101/2020.07.27.223081

    Figure Lengend Snippet: Nuclear transport factors are be introduced to nuclear periphery using digitonin permeabilization of the plasma membrane. A) Cells were incubated with R-phycoerythrin after the digitonin permeabilization protocol was performed with (left) 0 µg/mL digitonin, for which no R-phycoerythrin is detected in the cell, (middle) 34 µg/mL, for which R-phycoerythrin has entered the cytosol but is excluded from the nucleus, and (right) 100 µg/mL digitonin for which R-phycoerythrin is seen throughout the cytosol and nucleus. (Scale Bar = 10 µm). B-C) The Coomassie-stained gels show purified Ran, NTF2, Kap1-α, Kap1- β proteins with minimal contaminants. D) Cells were incubated with an ATP-regenerating system, Ran-GTP, NTF2, and an NLS-TD-Tomato cargo in the presence and absence of Kap1-α and Kap1- β . E) Amount of NLS-TD-Tomato accumulated in the nucleus was measured by quantifying the average fluorescence signal in the nucleus, as defined via Hoechst staining. (n=5 cells, error bars represent standard deviation). F-G) Removal of kaps was confirmed with quantitative IF. (Scale Bar = 10 µm; error bars represent standard deviation; n=5 cells) .

    Article Snippet: Both conditions contained the following base mix: 1.5 µM NLS-tdTomato, 0.1 mM GTP (ThermoFischer Scientific), 2 µM Ran, and 1 µM NTF2 in TB + pvp as described above.

    Techniques: Incubation, Staining, Purification, Fluorescence, Standard Deviation

    Cyclic dinucleotides activate the inflammasome in THP-1 cells and murine BMMs. ( A ) PMA-differentiated THP-1 cells (left panel) were treated with ATP (5 mM; 6 h) or transfected (Lipofectamine 2000; lpftm) with c-diGMP (cdG; 5 nmol; 6 h) or poly(dA:dT) (dA:dT; 5 μg/ml; 6 h). LPS-primed BMMs (right panel) were treated with ATP (5 mM; 6 h) or transfected (X-tremegene HP; Xtrm) with c-diGMP (10 nmol; 6 h), c-diAMP (10 nmol; 6 h), cGMP (10 nmol; 6 h) or GTP (10 nmol; 6 h). Supernatants were immunoblotted for immature IL-1β (pro-IL1β), active IL-1β (p17), immature caspase-1 (pro-Cas1) or active caspase-1 (Cas1, p20 and p10, respectively). ( B ) IL-1β levels were measured by ELISA (BD Biosciences) in 6 h supernatants of LPS-primed BMMs after the indicated treatment with c-diGMP or c-diAMP, as in A . ( C ) IL-1β levels were measured by ELISA in BMMs infected with IPTG-induced L. pneumophila ]. Graphs present means±standard error from three independent experiments. BMMs, bone marrow-derived macrophages; c-diAMP, 3′-5′ diadenylate; c-diGMP, 3′-5′ diguanylate; ELISA, enzyme-linked immunosorbent assay; MOI, multiplicity of infection.

    Journal: EMBO Reports

    Article Title: Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome

    doi: 10.1038/embor.2013.132

    Figure Lengend Snippet: Cyclic dinucleotides activate the inflammasome in THP-1 cells and murine BMMs. ( A ) PMA-differentiated THP-1 cells (left panel) were treated with ATP (5 mM; 6 h) or transfected (Lipofectamine 2000; lpftm) with c-diGMP (cdG; 5 nmol; 6 h) or poly(dA:dT) (dA:dT; 5 μg/ml; 6 h). LPS-primed BMMs (right panel) were treated with ATP (5 mM; 6 h) or transfected (X-tremegene HP; Xtrm) with c-diGMP (10 nmol; 6 h), c-diAMP (10 nmol; 6 h), cGMP (10 nmol; 6 h) or GTP (10 nmol; 6 h). Supernatants were immunoblotted for immature IL-1β (pro-IL1β), active IL-1β (p17), immature caspase-1 (pro-Cas1) or active caspase-1 (Cas1, p20 and p10, respectively). ( B ) IL-1β levels were measured by ELISA (BD Biosciences) in 6 h supernatants of LPS-primed BMMs after the indicated treatment with c-diGMP or c-diAMP, as in A . ( C ) IL-1β levels were measured by ELISA in BMMs infected with IPTG-induced L. pneumophila ]. Graphs present means±standard error from three independent experiments. BMMs, bone marrow-derived macrophages; c-diAMP, 3′-5′ diadenylate; c-diGMP, 3′-5′ diguanylate; ELISA, enzyme-linked immunosorbent assay; MOI, multiplicity of infection.

    Article Snippet: THP-1 cells were primed with phorbol myristate acetate (PMA; 100 nM, over night; Sigma-Aldrich) and BMMs were primed with LPS (1 μg/ml, 3 h; E. coli ; type 025:86; Sigma-Aldrich) before stimulation with ATP (Sigma-Aldrich), MSU (Enzo Life Sciences, Farmingdale, NY), crystalline silica (Min-U-Sil 5, U.S. Silica, Berkeley Springs, WV), S. typhimurium (see online), L. pneumophila (see online; [ ]), or transfection with synthetic c-diGMP or c-diAMP ( > 99% pure; [ ]), cGMP (Enzo Life Sciences), GTP (Sigma-Aldrich) or poly(dA:dT) (Sigma-Aldrich), as indicated.

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Infection, Derivative Assay

    Mutational analyses of basic residues in cpFtsY. A, representative stimulated GTPase reactions of cpSRP54 with wild-type cpFtsY ( black ) and mutants cpFtsY(K191D/K193D) ( blue ), cpFtsY(K203D/R204D) ( purple ), and cpFtsY(K234D/K235D) ( green . B, basal GTP hydrolysis rate constants for wild-type and mutant cpFtsY, determined as described under “Experimental Procedures.” C, fluorescence emission spectra for acrylodan-labeled cpSRP54 by itself (○) and in the presence of wild-type cpFtsY (●) or mutant cpFtsY(K203D/R204D) ( purple ).

    Journal: The Journal of Biological Chemistry

    Article Title: Co-evolution of Two GTPases Enables Efficient Protein Targeting in an RNA-less Chloroplast Signal Recognition Particle Pathway *

    doi: 10.1074/jbc.M116.752931

    Figure Lengend Snippet: Mutational analyses of basic residues in cpFtsY. A, representative stimulated GTPase reactions of cpSRP54 with wild-type cpFtsY ( black ) and mutants cpFtsY(K191D/K193D) ( blue ), cpFtsY(K203D/R204D) ( purple ), and cpFtsY(K234D/K235D) ( green . B, basal GTP hydrolysis rate constants for wild-type and mutant cpFtsY, determined as described under “Experimental Procedures.” C, fluorescence emission spectra for acrylodan-labeled cpSRP54 by itself (○) and in the presence of wild-type cpFtsY (●) or mutant cpFtsY(K203D/R204D) ( purple ).

    Article Snippet: To form the GTP-bound cpSRP54·cpFtsY or cpSRP54-NG·cpFtsY complex, 2 m m GTP (Sigma) was used; GDP released during the course of the reaction was minimal, as explained previously ( ).

    Techniques: Mutagenesis, Fluorescence, Labeling

    Effects of point mutations on nucleotide-binding motifs of KaiC and bioluminescence rhythm of P kaiBC . ( A ) Point mutations introduced to nucleotide-binding motifs (P loops and DXXG motifs) of KaiC are illustrated. The amino acid (aa) sequences of these motifs and point mutations are designated by their one-letter codes. Tandem duplicated domains of KaiC, N-terminal and C-terminal halves, are indicated as CI (amino acids 1–250) and CII (amino acids 251–519), respectively. Locations of P loops, DXXG motifs, Walker's motif Bs, and putative catalytic carboxylates are illustrated. Conserved amino acids of each motif are shown in boldface, and X represents any residue. ( B–D ) In vitro nucleotide binding assay ( Left ) and bioluminescence profiles for P kaiBC promoter activity ( Right ). 35 S-labeled KaiC was incubated with ATP agarose (lane 3), GTP agarose (lane 4), or control agarose (lane 2) resin. Input controls are shown in lane 1. Autoradiograms for 35 S-labeled KaiC are shown. Arrowheads indicate the position of the KaiC band (≈57 kDa). For monitoring bioluminescence rhythm, cells were grown to give 30–70 colonies 0.2 mm in diameter under LL conditions. After a 12-h dark treatment, the bioluminescence from each dish was measured with a photomultiplier tube. Bioluminescence intensity per colony was normalized to 100 by the peak value of the first day for wild-type cells ( y axes). ( B ) Binding to wild-type (WT) KaiC and bioluminescence from wild-type cells. ( C ) Lysine-to-histidine substitution in P loop 1 (K52H). ( D ) Lysine-to-histidine substitution in P loop 2 (K294H).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria

    doi:

    Figure Lengend Snippet: Effects of point mutations on nucleotide-binding motifs of KaiC and bioluminescence rhythm of P kaiBC . ( A ) Point mutations introduced to nucleotide-binding motifs (P loops and DXXG motifs) of KaiC are illustrated. The amino acid (aa) sequences of these motifs and point mutations are designated by their one-letter codes. Tandem duplicated domains of KaiC, N-terminal and C-terminal halves, are indicated as CI (amino acids 1–250) and CII (amino acids 251–519), respectively. Locations of P loops, DXXG motifs, Walker's motif Bs, and putative catalytic carboxylates are illustrated. Conserved amino acids of each motif are shown in boldface, and X represents any residue. ( B–D ) In vitro nucleotide binding assay ( Left ) and bioluminescence profiles for P kaiBC promoter activity ( Right ). 35 S-labeled KaiC was incubated with ATP agarose (lane 3), GTP agarose (lane 4), or control agarose (lane 2) resin. Input controls are shown in lane 1. Autoradiograms for 35 S-labeled KaiC are shown. Arrowheads indicate the position of the KaiC band (≈57 kDa). For monitoring bioluminescence rhythm, cells were grown to give 30–70 colonies 0.2 mm in diameter under LL conditions. After a 12-h dark treatment, the bioluminescence from each dish was measured with a photomultiplier tube. Bioluminescence intensity per colony was normalized to 100 by the peak value of the first day for wild-type cells ( y axes). ( B ) Binding to wild-type (WT) KaiC and bioluminescence from wild-type cells. ( C ) Lysine-to-histidine substitution in P loop 1 (K52H). ( D ) Lysine-to-histidine substitution in P loop 2 (K294H).

    Article Snippet: A fraction of the radioactivity (3.0 × 105 cpm) of the in vitro translation reaction mixture was diluted to 450 μl with binding buffer [20 mM Tris⋅HCl, pH 7.0/150 mM NaCl/5 mM MgCl2 /0.1% (vol/vol) Triton X-100] and incubated with 50 μl of ATP-agarose or GTP-agarose beads (2.2 μmol/ml; Sigma) at room temperature for 30 min.

    Techniques: Binding Assay, In Vitro, Activity Assay, Labeling, Incubation

    Analysis of functionally active pol III transcription complexes separated by a glycerol gradient. Several identical transcription reactions were performed in a volume of 25 µl each as described in Materials and Methods. After incubation they were pooled. Aliquots of 200 µl from this pool (corresponding to eight transcription reactions) were applied to a 4.2 ml glycerol gradient and centrifuged for 3 h at 50 000 r.p.m. in an SW 60 rotor. After centrifugation, fractions of 350 µl were collected. The corresponding fractions from six parallel gradients were pooled. ( A ) In vitro transcription. The fractions from the gradients and the load fraction were incubated with nucleotides, [α- 32 P]GTP and RNase Block as described above. No additional DNA was added. After incubation, the RNA in the samples was extracted as described above and loaded onto a 6% denaturing polyacrylamide gel. Lane 1, 25 µl load fraction; lanes 2–13, 50 µl glycerol gradient fractions. Fraction 1 is the top fraction ( B ) Western blot. The remaining pooled fractions were treated with Strataclean Resin (Stratagene) and loaded onto a 12.5% SDS gel. After blotting, the membrane was incubated with monoclonal anti-TBP antibodies and subsequently incubated with [ 125 I]anti-mouse antibodies as secondary antibodies. The membrane was analysed with a phosphorimager. The membrane was not stripped, but incubated with a mixture of SW5 and 3B9 antibodies and subsequently incubated with 125 I-labelled anti-mouse antibodies. The final analysis was performed with a phosphorimager and by autoradiography. Note that the band in fraction 8 at the height of the La signal was already visible after TBP incubation (data not shown). It is presumably an artefact due to the second antibody. Lane 1, 160 µl of load fraction; lanes 2–13, 1.85 ml glycerol gradient fractions; lane 14, 5 µl IIIBβ; lane 15, 5 µl of a La fraction, purified from PCA (20 ng La/µl). ( C ) In vitro transcription. The in vitro transcription was performed as described in (A) using a mixture of fractions 8 and 9 of a typical gradient. To ensure single round conditions in lanes 6–10, heparin was added at a final concentration of 600 µg/ml. Lanes 1–5, incubation without heparin for 2, 5, 10, 20 and 40 min; lanes 6–10, incubation with heparin for 2, 5, 10, 20 and 40 min. ( D ) Multiple round transcription of fractions 8 and 9 for 60 min. The gel was autoradiographed for a shorter time period as in (C). BSA (10 µg) was added to the reaction in order to avoid any protein effects. Lane 1, no additional La; lanes 2–4: addition of 15, 30 or 60 ng recombinant La.

    Journal: Nucleic Acids Research

    Article Title: Transcription efficiency of human polymerase III genes in vitro does not depend on the RNP-forming autoantigen La

    doi:

    Figure Lengend Snippet: Analysis of functionally active pol III transcription complexes separated by a glycerol gradient. Several identical transcription reactions were performed in a volume of 25 µl each as described in Materials and Methods. After incubation they were pooled. Aliquots of 200 µl from this pool (corresponding to eight transcription reactions) were applied to a 4.2 ml glycerol gradient and centrifuged for 3 h at 50 000 r.p.m. in an SW 60 rotor. After centrifugation, fractions of 350 µl were collected. The corresponding fractions from six parallel gradients were pooled. ( A ) In vitro transcription. The fractions from the gradients and the load fraction were incubated with nucleotides, [α- 32 P]GTP and RNase Block as described above. No additional DNA was added. After incubation, the RNA in the samples was extracted as described above and loaded onto a 6% denaturing polyacrylamide gel. Lane 1, 25 µl load fraction; lanes 2–13, 50 µl glycerol gradient fractions. Fraction 1 is the top fraction ( B ) Western blot. The remaining pooled fractions were treated with Strataclean Resin (Stratagene) and loaded onto a 12.5% SDS gel. After blotting, the membrane was incubated with monoclonal anti-TBP antibodies and subsequently incubated with [ 125 I]anti-mouse antibodies as secondary antibodies. The membrane was analysed with a phosphorimager. The membrane was not stripped, but incubated with a mixture of SW5 and 3B9 antibodies and subsequently incubated with 125 I-labelled anti-mouse antibodies. The final analysis was performed with a phosphorimager and by autoradiography. Note that the band in fraction 8 at the height of the La signal was already visible after TBP incubation (data not shown). It is presumably an artefact due to the second antibody. Lane 1, 160 µl of load fraction; lanes 2–13, 1.85 ml glycerol gradient fractions; lane 14, 5 µl IIIBβ; lane 15, 5 µl of a La fraction, purified from PCA (20 ng La/µl). ( C ) In vitro transcription. The in vitro transcription was performed as described in (A) using a mixture of fractions 8 and 9 of a typical gradient. To ensure single round conditions in lanes 6–10, heparin was added at a final concentration of 600 µg/ml. Lanes 1–5, incubation without heparin for 2, 5, 10, 20 and 40 min; lanes 6–10, incubation with heparin for 2, 5, 10, 20 and 40 min. ( D ) Multiple round transcription of fractions 8 and 9 for 60 min. The gel was autoradiographed for a shorter time period as in (C). BSA (10 µg) was added to the reaction in order to avoid any protein effects. Lane 1, no additional La; lanes 2–4: addition of 15, 30 or 60 ng recombinant La.

    Article Snippet: Aliquots of 50 µl of the gradient fractions were mixed with nucleotides, 3 µCi [α-32 P]GTP (Amersham) and RNase Block and transcribed as described above in a total volume of 60 µl.

    Techniques: Incubation, Centrifugation, In Vitro, Blocking Assay, Western Blot, SDS-Gel, Autoradiography, Purification, Concentration Assay, Recombinant

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Journal: Journal of Clinical Investigation

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    doi: 10.1172/JCI200316432

    Figure Lengend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Article Snippet: Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([3 H]GTP, Amersham) and increasing amounts of 6-Thio-GTP (0–500 μM) followed by Rac1 and Ras pull-down through PAK and Raf RGD and analysis of [3 H]GTP-bound Rac1 or Ras by scintillation counting.

    Techniques: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    Shear stress-stimulated GTPase activity of reconstituted G proteins. ( A ) G protein reconstituted liposomes loaded with [γ- 32 P]GTP were passed through Sephadex G-50 to remove external GTP. GTPase was stimulated when vesicles were subjected to shear stress (0–30 dynes/cm 2 ) in a cone-and-plate viscometer (37°C, 1 min). This activity was attenuated by incorporation of cholesterol (24 mol %). ( Inset ) Mastoparan (900 μM) incubated with liposomes for 1 min (37°C) stimulated GTPase activity. ( B ) Affinity-purified G αq and G αi3 , reconstituted into liposomes along with their respective βγ subunits (immunoblots for α and β are shown in Inset ) were stimulated by shear stress. The values presented here are a mean ± SD of three measurements.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition

    doi:

    Figure Lengend Snippet: Shear stress-stimulated GTPase activity of reconstituted G proteins. ( A ) G protein reconstituted liposomes loaded with [γ- 32 P]GTP were passed through Sephadex G-50 to remove external GTP. GTPase was stimulated when vesicles were subjected to shear stress (0–30 dynes/cm 2 ) in a cone-and-plate viscometer (37°C, 1 min). This activity was attenuated by incorporation of cholesterol (24 mol %). ( Inset ) Mastoparan (900 μM) incubated with liposomes for 1 min (37°C) stimulated GTPase activity. ( B ) Affinity-purified G αq and G αi3 , reconstituted into liposomes along with their respective βγ subunits (immunoblots for α and β are shown in Inset ) were stimulated by shear stress. The values presented here are a mean ± SD of three measurements.

    Article Snippet: [γ-32 P]GTP (3,000 Ci/mmol; 1 Ci = 37 GBq) was from Amersham and 35 S-labeled GTP[γ-S] (GTP[γ-35 S]; ≈1,100–1,300 Ci/mmol) was from DuPont NEN.

    Techniques: Activity Assay, Incubation, Affinity Purification, Western Blot

    Capacity of GTP γ S to Inhibit PTH Radioligand Binding to Wild-Type and Mutant PTHRs in COS-7 Cell Membranes

    Journal:

    Article Title: Mechanisms of Ligand Binding to the Parathyroid Hormone (PTH)/PTH-Related Protein Receptor: Selectivity of a Modified PTH(1-15) Radioligand for GαS-Coupled Receptor Conformations

    doi: 10.1210/me.2005-0349

    Figure Lengend Snippet: Capacity of GTP γ S to Inhibit PTH Radioligand Binding to Wild-Type and Mutant PTHRs in COS-7 Cell Membranes

    Article Snippet: The dissociation phase was then initiated by the addition of a saturating concentration of the unlabeled ligand (1 × 10−6 m final concentration) with or without GTP γ S (Sigma-Aldrich Inc., St. Louis, MO) at a final concentration of 5 × 10−5 m .

    Techniques: Binding Assay, Mutagenesis

    Effect of GTP γ S on PTH Radioligand Binding to HKRK-B7 Cell Membranes

    Journal:

    Article Title: Mechanisms of Ligand Binding to the Parathyroid Hormone (PTH)/PTH-Related Protein Receptor: Selectivity of a Modified PTH(1-15) Radioligand for GαS-Coupled Receptor Conformations

    doi: 10.1210/me.2005-0349

    Figure Lengend Snippet: Effect of GTP γ S on PTH Radioligand Binding to HKRK-B7 Cell Membranes

    Article Snippet: The dissociation phase was then initiated by the addition of a saturating concentration of the unlabeled ligand (1 × 10−6 m final concentration) with or without GTP γ S (Sigma-Aldrich Inc., St. Louis, MO) at a final concentration of 5 × 10−5 m .

    Techniques: Binding Assay

    Organization of actin stress fiber, the association of actin with paxillin, and RhoA activity are decreased in BHDS lung fibroblasts. (A) Representative confocal images of phalloidin and paxillin staining in lung fibroblasts are shown; the results of BHDS lung fibroblasts isolated from patient 2 harboring c.769_777del TCC (exon 7) mutation are presented. The immunofluorescence staining was performed in both control ( n = 4) and BHDS ( n = 4: patients 2, 7, 10, and 11). Scale bars: 50 μm in each panel. (B) The levels of GTP ‐bound RhoA (Rho‐ GTP ) and Rho GTP /total RhoA ratio are decreased in BHDS lung fibroblasts ( n = 4: patients 2, 7, 10, and 11) as compared with those of control lung fibroblasts ( n = 4) (* P

    Journal: Physiological Reports

    Article Title: Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt–Hogg–Dubé syndrome. Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt–Hogg–Dubé syndrome

    doi: 10.14814/phy2.13025

    Figure Lengend Snippet: Organization of actin stress fiber, the association of actin with paxillin, and RhoA activity are decreased in BHDS lung fibroblasts. (A) Representative confocal images of phalloidin and paxillin staining in lung fibroblasts are shown; the results of BHDS lung fibroblasts isolated from patient 2 harboring c.769_777del TCC (exon 7) mutation are presented. The immunofluorescence staining was performed in both control ( n = 4) and BHDS ( n = 4: patients 2, 7, 10, and 11). Scale bars: 50 μm in each panel. (B) The levels of GTP ‐bound RhoA (Rho‐ GTP ) and Rho GTP /total RhoA ratio are decreased in BHDS lung fibroblasts ( n = 4: patients 2, 7, 10, and 11) as compared with those of control lung fibroblasts ( n = 4) (* P

    Article Snippet: Determination of the levels of active GTP‐bound RhoA and total RhoA The levels of active GTP‐bound RhoA and total RhoA were examined using the G‐LISA® Active RhoA Activation Assay Biochem kit (Cytoskeleton Inc., Denver, CO) and the Total RhoA ELISA Biochem Kit (Cytoskeleton Inc.), respectively, according to the manufacturer's instruction.

    Techniques: Activity Assay, Staining, Isolation, Mutagenesis, Immunofluorescence

    Levels of activated RhoA-GTP (A) and Rac1-GTP (B) for cells infected with various N. meningitidis isolates at an early time point (5 min) and a late time point (14 h). A total of 50 μg of total protein was assayed per sample ( n ≥ 3 replicates/sample).

    Journal: Infection and Immunity

    Article Title: Disease-Associated Neisseria meningitidis Isolates Inhibit Wound Repair in Respiratory Epithelial Cells in a Type IV Pilus-Independent Manner

    doi: 10.1128/IAI.02001-14

    Figure Lengend Snippet: Levels of activated RhoA-GTP (A) and Rac1-GTP (B) for cells infected with various N. meningitidis isolates at an early time point (5 min) and a late time point (14 h). A total of 50 μg of total protein was assayed per sample ( n ≥ 3 replicates/sample).

    Article Snippet: The amount of Rac1- and RhoA-GTP was measured using a G-LISA Rac1 and RhoA Biochem kit (absorbance based; Cytoskeleton, Inc.) according to manufacturer's directions.

    Techniques: Infection

    Full-length hAGO2 binds to m 7 GTP-Sepharose nonspecifically in in vitro pull-down experiments and at low levels, indicating background binding. ( A ) Pull-down experiments using recombinant GST-eIF4E and hexahistidine-SUMO-tagged hAGO2 MID domain with m

    Journal: EMBO Reports

    Article Title: Structural analysis of 5?-mRNA-cap interactions with the human AGO2 MID domain

    doi: 10.1038/embor.2011.48

    Figure Lengend Snippet: Full-length hAGO2 binds to m 7 GTP-Sepharose nonspecifically in in vitro pull-down experiments and at low levels, indicating background binding. ( A ) Pull-down experiments using recombinant GST-eIF4E and hexahistidine-SUMO-tagged hAGO2 MID domain with m

    Article Snippet: It should be noted that m7 GTP-Sepharose beads (from GE Healthcare) differ in their chemical conjugation from GMP/GTP-agarose (from Sigma) in that m7 GTP is linked to the matrix by the γ-phosphate, whereas both GMP and GTP are attached by either their 2′-OH or the 3′-OH of the ribose sugar.

    Techniques: In Vitro, Binding Assay, Recombinant

    Phosphorylation of eIF4E and 4E-BP1 is impaired in serum-starved cells. Hep G2 cells were grown in Eagle's MEM supplemented with 10% fetal calf serum (stimulated lanes) or cultured in serum-free media for 15 h (starved lanes). ( A ) Cap binding proteins were purified on m7 GTP-Sepharose 4B beads and electrophoresed through a 3.5–10 pH gradient isoelectric focussing gel. Western blotting was performed using α-eIF4E antisera. ( B ) Proteins from S10 or S100 cellular fractions prepared from Hep G2 cells grown under the indicated conditions were electrophoresed on a 15% polyacrylamide gel containing SDS. Phosphorylated 4E-BP1 binding protein was specifically detected by western blotting with antisera to the ser65 phosphorylated isoform.

    Journal: Nucleic Acids Research

    Article Title: Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

    doi: 10.1093/nar/gki169

    Figure Lengend Snippet: Phosphorylation of eIF4E and 4E-BP1 is impaired in serum-starved cells. Hep G2 cells were grown in Eagle's MEM supplemented with 10% fetal calf serum (stimulated lanes) or cultured in serum-free media for 15 h (starved lanes). ( A ) Cap binding proteins were purified on m7 GTP-Sepharose 4B beads and electrophoresed through a 3.5–10 pH gradient isoelectric focussing gel. Western blotting was performed using α-eIF4E antisera. ( B ) Proteins from S10 or S100 cellular fractions prepared from Hep G2 cells grown under the indicated conditions were electrophoresed on a 15% polyacrylamide gel containing SDS. Phosphorylated 4E-BP1 binding protein was specifically detected by western blotting with antisera to the ser65 phosphorylated isoform.

    Article Snippet: Aliquots (100 μg) of post 600 g supernatant were incubated with 30 μl m7 GTP-Sepharose (6 nmol ligand, GE Healthcare, Amersham Biosciences) for 15 min at 4°C.

    Techniques: Cell Culture, Binding Assay, Purification, Western Blot

    Serum starvation differentially affects the interaction of PARN and eIF4E with the mRNA 5′ cap. ( A ) Diagrammatic representation of the luciferase mRNA and its two variants used in this study. SL–Luc had a stem loop structure inserted into its 5′ UTR. Luc–Cox contained the COX4 3′ UTR attached to the luciferase opening reading frame. All constructs were capped and contained a 40 base adenosine 3′ tail. ( B ) The RNAs outlined in panel A were labelled exclusively at their 5′ cap structure and incubated in cell extracts derived from serum-stimulated (stimulated lanes) or serum-starved cells (starved lanes). Reactions mixtures were irradiated with UV light to covalently cross-link closely associated proteins and treated with RNase. Samples were then immunoprecipitated using α-PARN (top half) or α-eIF4E (bottom half) antisera. Immunoprecipitated proteins that were cross linked to the radioactive cap were separated on polyacrylamide gels containing SDS, visualized by phosphorimaging and analysed using ImageQuant software. ( C ) Post 600 g supernatants prepared from cells grown under serum-starvation conditions (starved lanes) or in the presence of 10% serum (stimulated lanes) were incubated with m7 GTP-Sepharose beads. Beads were washed extensively and bound proteins were electrophoresed on a 10% polyacrylamide gel with SDS. Western blots were performed to assess the levels of bound PARN (top half) and eIF4E (bottom half) using specific antisera.

    Journal: Nucleic Acids Research

    Article Title: Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

    doi: 10.1093/nar/gki169

    Figure Lengend Snippet: Serum starvation differentially affects the interaction of PARN and eIF4E with the mRNA 5′ cap. ( A ) Diagrammatic representation of the luciferase mRNA and its two variants used in this study. SL–Luc had a stem loop structure inserted into its 5′ UTR. Luc–Cox contained the COX4 3′ UTR attached to the luciferase opening reading frame. All constructs were capped and contained a 40 base adenosine 3′ tail. ( B ) The RNAs outlined in panel A were labelled exclusively at their 5′ cap structure and incubated in cell extracts derived from serum-stimulated (stimulated lanes) or serum-starved cells (starved lanes). Reactions mixtures were irradiated with UV light to covalently cross-link closely associated proteins and treated with RNase. Samples were then immunoprecipitated using α-PARN (top half) or α-eIF4E (bottom half) antisera. Immunoprecipitated proteins that were cross linked to the radioactive cap were separated on polyacrylamide gels containing SDS, visualized by phosphorimaging and analysed using ImageQuant software. ( C ) Post 600 g supernatants prepared from cells grown under serum-starvation conditions (starved lanes) or in the presence of 10% serum (stimulated lanes) were incubated with m7 GTP-Sepharose beads. Beads were washed extensively and bound proteins were electrophoresed on a 10% polyacrylamide gel with SDS. Western blots were performed to assess the levels of bound PARN (top half) and eIF4E (bottom half) using specific antisera.

    Article Snippet: Aliquots (100 μg) of post 600 g supernatant were incubated with 30 μl m7 GTP-Sepharose (6 nmol ligand, GE Healthcare, Amersham Biosciences) for 15 min at 4°C.

    Techniques: Luciferase, Construct, Incubation, Derivative Assay, Irradiation, Immunoprecipitation, Software, Western Blot

    Increased binding of the translation initiation factor eIF4G to a synthetic 7-methyl-GTP (7MeGTP) mRNA cap structure in HPV16 E6-expressing RKO cell lysates, which is sensitive to rapamycin treatment. Control and HPV16 E6-expressing RKO cells were treated with dimethyl sulfoxide (DMSO) or 100 nM rapamycin (Rap) for 1 h prior to lysis. Cap binding assays were performed as described in Materials and Methods. Levels of eIF4G in a 50-μg sample, representing 25% of the cap-binding reaction, together with an actin blot, are shown in the top panel (Input). Blot results for cap-bound eIF4G are shown in the bottom panel.

    Journal: Journal of Virology

    Article Title: The Human Papillomavirus Type 16 E6 Oncoprotein Activates mTORC1 Signaling and Increases Protein Synthesis ▿The Human Papillomavirus Type 16 E6 Oncoprotein Activates mTORC1 Signaling and Increases Protein Synthesis ▿ †

    doi: 10.1128/JVI.00974-10

    Figure Lengend Snippet: Increased binding of the translation initiation factor eIF4G to a synthetic 7-methyl-GTP (7MeGTP) mRNA cap structure in HPV16 E6-expressing RKO cell lysates, which is sensitive to rapamycin treatment. Control and HPV16 E6-expressing RKO cells were treated with dimethyl sulfoxide (DMSO) or 100 nM rapamycin (Rap) for 1 h prior to lysis. Cap binding assays were performed as described in Materials and Methods. Levels of eIF4G in a 50-μg sample, representing 25% of the cap-binding reaction, together with an actin blot, are shown in the top panel (Input). Blot results for cap-bound eIF4G are shown in the bottom panel.

    Article Snippet: Lysates from RKO E6 and RKO control cells were incubated with 7-methyl-GTP-Sepharose, and association of initiation factor eIF4G was evaluated by Western blotting.

    Techniques: Binding Assay, Expressing, Lysis

    DOCK10 is a GEF for Rac1 and Cdc42 in vitro and in cells. (A, B) In vitro GEF assay performed on Cdc42 (A) and Rac1 (B), using GST-DHR2 domains of DOCK9, DOCK10, and DOCK11. GST-Dbl and GST-Trio GEF1 were used as positive control GEFs for Cdc42 and Rac1, respectively, whereas GST alone was used as negative control. Results are expressed as relative fluorescence units (RFU) vs. time. (C–E) Active GTPase pull-down assays. GTP-loaded Cdc42 (C) or GTP-loaded Rac1 (D, E) were pulled down from protein lysates of HEK293 cells expressing wild-type pEGFP-DOCK9, -10 or -11 constructs or DOCK10 GD. GFP vector was used as a negative control. Shown are representative experiments. GTP-bound and total Cdc42 or Rac1 were detected by Western blot, using anti-Cdc42 and anti-Rac1 antibodies, respectively. Protein expression in the cell lysates was verified by immunoblotting with an anti-GFP antibody. Histograms underneath each Western blot show the quantification of the corresponding GTPase activation assays. The ratio of GTP-Rac1 (or Cdc42) over total Rac1 (or Cdc42) was calculated from at least three independent experiments. Error bars indicate SEM, ** p

    Journal: Molecular Biology of the Cell

    Article Title: The RhoGEF DOCK10 is essential for dendritic spine morphogenesis

    doi: 10.1091/mbc.E14-08-1310

    Figure Lengend Snippet: DOCK10 is a GEF for Rac1 and Cdc42 in vitro and in cells. (A, B) In vitro GEF assay performed on Cdc42 (A) and Rac1 (B), using GST-DHR2 domains of DOCK9, DOCK10, and DOCK11. GST-Dbl and GST-Trio GEF1 were used as positive control GEFs for Cdc42 and Rac1, respectively, whereas GST alone was used as negative control. Results are expressed as relative fluorescence units (RFU) vs. time. (C–E) Active GTPase pull-down assays. GTP-loaded Cdc42 (C) or GTP-loaded Rac1 (D, E) were pulled down from protein lysates of HEK293 cells expressing wild-type pEGFP-DOCK9, -10 or -11 constructs or DOCK10 GD. GFP vector was used as a negative control. Shown are representative experiments. GTP-bound and total Cdc42 or Rac1 were detected by Western blot, using anti-Cdc42 and anti-Rac1 antibodies, respectively. Protein expression in the cell lysates was verified by immunoblotting with an anti-GFP antibody. Histograms underneath each Western blot show the quantification of the corresponding GTPase activation assays. The ratio of GTP-Rac1 (or Cdc42) over total Rac1 (or Cdc42) was calculated from at least three independent experiments. Error bars indicate SEM, ** p

    Article Snippet: In vitro GEF assays Fluorescence-based in vitro guanine-nucleotide exchange assays were performed using Mant-GTP (Molecular Probes, Life Technologies, St-Aubin, France) in an FLX 800 microplate fluorescence reader (BioTek Instruments, Colmar, France) at 25°C, as described ( ).

    Techniques: In Vitro, GEF Assay, Positive Control, Negative Control, Fluorescence, Expressing, Construct, Plasmid Preparation, Western Blot, Activation Assay

    Combination of CTP and GTP with 2CMC in the Huh7-p6-luc model (A) and the Huh7-p6 model (B). The cells were treated with 2CMC, CTP or GTP, alone or in combination for 72 h before measurement of luciferase activity. Data are the mean ± SEM of four to six independent experiments. *, P

    Journal: Archives of Virology

    Article Title: Nucleoside analogue 2’-C-methylcytidine inhibits hepatitis E virus replication but antagonizes ribavirin

    doi: 10.1007/s00705-017-3444-8

    Figure Lengend Snippet: Combination of CTP and GTP with 2CMC in the Huh7-p6-luc model (A) and the Huh7-p6 model (B). The cells were treated with 2CMC, CTP or GTP, alone or in combination for 72 h before measurement of luciferase activity. Data are the mean ± SEM of four to six independent experiments. *, P

    Article Snippet: Reagents and antibodies 2CMC, RBV, guanosine triphosphate (GTP) and cytidine 5’-triphosphate (CTP) were purchased from Sigma-Aldrich and were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO).

    Techniques: Luciferase, Activity Assay

    Tubulin-directed activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 P]GTP. GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Mutations of Oncoprotein 18/Stathmin Identify Tubulin-Directed Regulatory Activities Distinct from Tubulin Association

    doi:

    Figure Lengend Snippet: Tubulin-directed activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 P]GTP. GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.

    Article Snippet: The protein concentration of the extracts was adjusted to 1 mg/ml, and the extracts were incubated with GTP (1 mM)–bovine tubulin (10 μM) (Cytoskeleton, Denver, Colo.) in PEM buffer (pH 6.8).

    Techniques: Incubation, Concentration Assay, Activity Assay

    Modulation of nocodazole-stimulated tubulin GTPase activity by wt and mutated Op18. Tubulin (5 μM) was incubated at 37°C in the presence of [γ- 32 P]GTP in the absence (open bars) or presence (striped bars) of nocodazole (33 μM). The indicated Op18 derivative (16 μM) was also included in the reaction mixtures. The means of duplicate determinations of hydrolyzed GTP, after 120 min of incubation, are shown. Data are representative for at least two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Mutations of Oncoprotein 18/Stathmin Identify Tubulin-Directed Regulatory Activities Distinct from Tubulin Association

    doi:

    Figure Lengend Snippet: Modulation of nocodazole-stimulated tubulin GTPase activity by wt and mutated Op18. Tubulin (5 μM) was incubated at 37°C in the presence of [γ- 32 P]GTP in the absence (open bars) or presence (striped bars) of nocodazole (33 μM). The indicated Op18 derivative (16 μM) was also included in the reaction mixtures. The means of duplicate determinations of hydrolyzed GTP, after 120 min of incubation, are shown. Data are representative for at least two independent experiments.

    Article Snippet: The protein concentration of the extracts was adjusted to 1 mg/ml, and the extracts were incubated with GTP (1 mM)–bovine tubulin (10 μM) (Cytoskeleton, Denver, Colo.) in PEM buffer (pH 6.8).

    Techniques: Activity Assay, Incubation

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Journal: Journal of Clinical Investigation

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    doi: 10.1172/JCI200316432

    Figure Lengend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Article Snippet: To analyze the capacity of 6-Thio-GTP to bind to Rac1 or Ras, chemically synthesized 6-Thio-GTP (obtained from Jena Bioscience, Jena, Germany) was used.

    Techniques: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

    Journal: Physiological Reports

    Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

    doi: 10.14814/phy2.12442

    Figure Lengend Snippet: HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

    Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

    Techniques: Activation Assay, Mouse Assay, Incubation, Staining

    Cytohesins are required to promote HGF-induced activation of Rac1 and Arf6 in injured kidney. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and stained either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

    Journal: Physiological Reports

    Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

    doi: 10.14814/phy2.12442

    Figure Lengend Snippet: Cytohesins are required to promote HGF-induced activation of Rac1 and Arf6 in injured kidney. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and stained either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

    Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

    Techniques: Activation Assay, Mouse Assay, Staining

    Cytohesin-dependent Arf6 to Rac1 signaling module is required to promote the HGF-stimulated recovery of damaged kidney. Treatment of IRI kidneys with HGF activates the HGF receptor c-met, resulting in a signaling cascade event that leads to activation of the Arf GEFs cytohesins. Active cytohesins promote Arf6 to exchange its bound GDP molecule for GTP, resulting in its activation. GTP-bound Arf6 thus activates Rac1. Activation of Rac1 induces epithelial cell migration to the bare areas of the tubules, promoting damaged kidney recovery.

    Journal: Physiological Reports

    Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

    doi: 10.14814/phy2.12442

    Figure Lengend Snippet: Cytohesin-dependent Arf6 to Rac1 signaling module is required to promote the HGF-stimulated recovery of damaged kidney. Treatment of IRI kidneys with HGF activates the HGF receptor c-met, resulting in a signaling cascade event that leads to activation of the Arf GEFs cytohesins. Active cytohesins promote Arf6 to exchange its bound GDP molecule for GTP, resulting in its activation. GTP-bound Arf6 thus activates Rac1. Activation of Rac1 induces epithelial cell migration to the bare areas of the tubules, promoting damaged kidney recovery.

    Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

    Techniques: Activation Assay, Migration

    HGF induces the activation of Rac1 and Arf6 in damaged kidneys early after reperfusion. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both 24 h after reperfusion. Injured kidneys were then harvested 24 h after treatment, cryosectioned (10 mm), and stained either against GTP-Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

    Journal: Physiological Reports

    Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

    doi: 10.14814/phy2.12442

    Figure Lengend Snippet: HGF induces the activation of Rac1 and Arf6 in damaged kidneys early after reperfusion. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both 24 h after reperfusion. Injured kidneys were then harvested 24 h after treatment, cryosectioned (10 mm), and stained either against GTP-Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

    Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

    Techniques: Activation Assay, Mouse Assay, Staining

    pUL69 associates with polysomes. MRC5 cells were harvested at 72 h after infection with TN wt . ( A ) Polysome preparation and analysis. Cytoplasmic extracts of infected MRC5 cells were prepared and fractionated by centrifugation on a 10 to 50% sucrose gradient. The bottom of gradient is to the left, and fractions 1 to 4 correspond to polysomes. ( Top ) UV absorbance profile of gradient fractions; ( Middle ) Analysis of total RNA present in gradient fractions by electrophoresis in an agarose gel; ( Bottom ) Western blot analysis of proteins in gradient fractions using antibodies for pUL69 and PABPC1. ( B ) Disruption of polysomes displaces pUL69 from rapidly sedimenting fractions. Cytoplasmic extracts were treated with EDTA (+) or left untreated (−) and subjected to centrifugation; proteins in fractions 1 to 4 containing polysomes were precipitated and analyzed by Western blot by using antibodies for pUL69, PABPC1 and eIF4A1. T, total cytoplasmic extract. ( C ) pUL69 interacts with viral mRNAs. Cytoplasmic extracts were subjected to immunoprecipitation by using an antibody to pUL69 pUL84 or they were mixed with protein A/G Sepharose without first receiving a primary antibody (No Ab). mRNA was isolated from the immunoprecipitates and quantified by RT-PCR using sequence-specific primers.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

    doi: 10.1073/pnas.0914856107

    Figure Lengend Snippet: pUL69 associates with polysomes. MRC5 cells were harvested at 72 h after infection with TN wt . ( A ) Polysome preparation and analysis. Cytoplasmic extracts of infected MRC5 cells were prepared and fractionated by centrifugation on a 10 to 50% sucrose gradient. The bottom of gradient is to the left, and fractions 1 to 4 correspond to polysomes. ( Top ) UV absorbance profile of gradient fractions; ( Middle ) Analysis of total RNA present in gradient fractions by electrophoresis in an agarose gel; ( Bottom ) Western blot analysis of proteins in gradient fractions using antibodies for pUL69 and PABPC1. ( B ) Disruption of polysomes displaces pUL69 from rapidly sedimenting fractions. Cytoplasmic extracts were treated with EDTA (+) or left untreated (−) and subjected to centrifugation; proteins in fractions 1 to 4 containing polysomes were precipitated and analyzed by Western blot by using antibodies for pUL69, PABPC1 and eIF4A1. T, total cytoplasmic extract. ( C ) pUL69 interacts with viral mRNAs. Cytoplasmic extracts were subjected to immunoprecipitation by using an antibody to pUL69 pUL84 or they were mixed with protein A/G Sepharose without first receiving a primary antibody (No Ab). mRNA was isolated from the immunoprecipitates and quantified by RT-PCR using sequence-specific primers.

    Article Snippet: Chromatography was as described previously ( ): 5 × 106 infected fibroblasts were dissolved in 1-mL lysis buffer [50 mM hepes, pH 7.4; 150 mM NaCl; 2mM EDTA; 2 mM Na3 VO4 ; 25 mM glycerophosphate; mini protease inhibitor mixture (Roche Applied Science); 0.5% Nonidet P-40], the lysate was precleared for 20 min at 4 °C with 30 μL (settled bed volume) Sepharose 4B, and then incubated for 1 h at 4°C with 50 μL (settled bed volume) m7 GTP Sepharose 4B (GE Healthcare).

    Techniques: Infection, Centrifugation, Electrophoresis, Agarose Gel Electrophoresis, Western Blot, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Sequencing

    UL69 excludes 4EBP1 from the cap-binding complex. Cells were mock infected (M) or infected with TN wt (WT) or TN sub UL69 (UL69 − ). ( A ) m 7 GTP Sepharose chromatography. At 72 hpi, extracts were prepared and proteins bound to m 7 GTP Sepharose were analyzed by Western blot using antibodies to the indicated proteins. ( B ) Specificity of m 7 GTP Sepharose interactions. ( Left ) Experiment was as described in A . ( Right ) Total cell extracts were analyzed by Western blot. α-tub, α-tubulin.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

    doi: 10.1073/pnas.0914856107

    Figure Lengend Snippet: UL69 excludes 4EBP1 from the cap-binding complex. Cells were mock infected (M) or infected with TN wt (WT) or TN sub UL69 (UL69 − ). ( A ) m 7 GTP Sepharose chromatography. At 72 hpi, extracts were prepared and proteins bound to m 7 GTP Sepharose were analyzed by Western blot using antibodies to the indicated proteins. ( B ) Specificity of m 7 GTP Sepharose interactions. ( Left ) Experiment was as described in A . ( Right ) Total cell extracts were analyzed by Western blot. α-tub, α-tubulin.

    Article Snippet: Chromatography was as described previously ( ): 5 × 106 infected fibroblasts were dissolved in 1-mL lysis buffer [50 mM hepes, pH 7.4; 150 mM NaCl; 2mM EDTA; 2 mM Na3 VO4 ; 25 mM glycerophosphate; mini protease inhibitor mixture (Roche Applied Science); 0.5% Nonidet P-40], the lysate was precleared for 20 min at 4 °C with 30 μL (settled bed volume) Sepharose 4B, and then incubated for 1 h at 4°C with 50 μL (settled bed volume) m7 GTP Sepharose 4B (GE Healthcare).

    Techniques: Binding Assay, Infection, Chromatography, Western Blot

    Proposed model. ( A ) Model depicting Arfrp1- and AP-1-mediated TGN sorting of Vangl2. Arfrp1 is recruited to TGN membranes upon GTP binding, possibly mediated by a TGN localized GEF. Subsequently, GTP-bound Arfrp1 recruits AP-1 to TGN membranes. GTP-bound Arfrp1 also promotes an open conformation of AP-1 that directly interacts with the tyrosine motif on Vangl2 cytosolic domain, thereby enriching Vangl2 in budding vesicles. Binding of Vangl2 cytosolic domain to AP-1, in turn, stabilizes the membrane association of AP-1 to allow sufficient time for AP-1 polymerization (possibly with clathrin as a coat outer layer) and vesicle budding. This model is consistent with reports showing that tyrosine sorting motifs promote membrane recruitment of AP-1 mediated by Arf1 ( Crottet et al., 2002 ; Lee et al., 2008 ). ( B ) The asymmetrically localized PCP signaling molecules, including Vangl2 and Frizzled 6, are sorted by different sorting machineries for export from the TGN. Differential TGN sorting and polarized trafficking of these signaling receptors may contribute to their asymmetric distribution and the laterally polarized organization of epithelial cells. DOI: http://dx.doi.org/10.7554/eLife.00160.017

    Journal: eLife

    Article Title: A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network

    doi: 10.7554/eLife.00160

    Figure Lengend Snippet: Proposed model. ( A ) Model depicting Arfrp1- and AP-1-mediated TGN sorting of Vangl2. Arfrp1 is recruited to TGN membranes upon GTP binding, possibly mediated by a TGN localized GEF. Subsequently, GTP-bound Arfrp1 recruits AP-1 to TGN membranes. GTP-bound Arfrp1 also promotes an open conformation of AP-1 that directly interacts with the tyrosine motif on Vangl2 cytosolic domain, thereby enriching Vangl2 in budding vesicles. Binding of Vangl2 cytosolic domain to AP-1, in turn, stabilizes the membrane association of AP-1 to allow sufficient time for AP-1 polymerization (possibly with clathrin as a coat outer layer) and vesicle budding. This model is consistent with reports showing that tyrosine sorting motifs promote membrane recruitment of AP-1 mediated by Arf1 ( Crottet et al., 2002 ; Lee et al., 2008 ). ( B ) The asymmetrically localized PCP signaling molecules, including Vangl2 and Frizzled 6, are sorted by different sorting machineries for export from the TGN. Differential TGN sorting and polarized trafficking of these signaling receptors may contribute to their asymmetric distribution and the laterally polarized organization of epithelial cells. DOI: http://dx.doi.org/10.7554/eLife.00160.017

    Article Snippet: Thank you for choosing to send your work entitled “A Novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network” for consideration at eLife.

    Techniques: Binding Assay